Development and evaluation of a real-time fluorescent quantitative polymerase chain reaction assay for detecting human Herpesvirus-8 viral load / 中华传染病杂志

ABSTRACT

Objective To establish a real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) for detection of human Herpesvirus-8 (HHV-8) viral load. Methods pMD19-T recombinant vectors inserted with an open reading frame (ORF) 26 of HHV-8 or β-actin gene were constructed respectively. A sensitive RT-qPCR method was established and optimized. The effectivity of the method was evaluated by determining the HHV-8 viral loads in 30 (formalin fixed, paraffinised)biopsy samples of Kaposi's sarcoma. Results The key factors for optimizing the method included anneal temperature and extension. The standard curve showed that the Ct value of ORF26 and β-actin had a good linear relationship (r2 ＞0.990) with the standard samples. The melt curve and electrophoresis showed the specificity of our study. The sensitivity of this method was very high and the detection rate could reach 100%. The viral loads were significantly higher in patients with classic Kaposi's sarcoma compared to patients with acquired immunodeficiency syndrome-associated Kaposi's sarcoma(69.18 va 8. 63, x2 =7.950,P=0.005).Conclusions The established RT-qPCR method is highly sensitive, which can be used as a routine assay for detecting HHV-8.This system offers a good platform for diagnosing other causative organism.